Effect of the mec1 and tel1 mutations on sensitivity to phleomycin. Wild-type (KSC1178), mec1Δ (KSC1186), tel1Δ (KSC1368), mec1-81 (KSC1400), mec1-81 tel1Δ (KSC1402) cells were grown to log phase and cell cultures were serially diluted, spotted on the YEPD plates with or without 5 µg/ml phleomycin.

Effect of the mec1Δ and tel1Δ mutations on phleomycin-induced Rad53 phosphorylation. Wild-type (KSC1178), mec1Δ (KSC1186) and tel1Δ (KSC1368) cells carrying YCp-RAD53-HA were untreated or treated with 5 or 50 µg/ml phleomycin for 120 min. Cell extracts were then subjected to immunoblotting analysis with anti-HA antibodies as described in Materials and Methods.

Effect of the mec1Δ and tel1Δ mutations on Xrs2 phosphorylation after phleomycin and MMS treatment in S phase. Wild-type (KSC1178), mec1Δ (KSC1186) and tel1Δ (KSC1368) cells carrying YCpT-XRS2-HA were synchronized with α-factor in G1 and released into 25 µg/ml phleomycin or 0.1% MMS as in Figure 2. Aliquots of cells were collected at the indicated times after release from α-factor treatment and subjected to immunoblotting.

Effect of the mec1Δ and tel1Δ mutations on the MMS-induced Rad53 phosphorylation in S phase. Wild-type (KSC1178), mec1Δ (KSC1186) and tel1Δ (KSC1368) cells carrying YCp-RAD53-HA were synchronized with α-factor in G1 and released in 0.1% MMS as in Figure 2. Aliquots of cells were collected at the indicated times after release from α-factor treatment and subjected to immunoblotting (A) and flow cytometric analyses (B) as in Figure 2.

Checkpoint responses after phleomycin treatment in G2/M phase. (A) Cell-cycle progression in G2/M phase after phleomycin treatment. Cells were synchronized with nocodazole in G2/M and further treated with 50 µg/ml phleomycin for 60 min. At the indicated times after release of phleomycin-treated (+PM) and untreated (–PM) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (B) Rad53 phosphorylation after phleomycin treatment in G2/M. Cells carrying YCp-RAD53-HA were arrested in G2/M with nocodazole and untreated or treated with 50 µg/ml phleomycin maintaining the cell-cycle arrest. Cells collected at the indicated time were subjected to immunoblotting analysis with anti-HA antibodies. Strains used are wild-type (KSC1178), mec1Δ (KSC1186) and tel1Δ (KSC1368).

Checkpoint responses after phleomycin treatment in G1 phase. (A) Cell-cycle progression at the G1/S transition after phleomycin treatment. Cells were synchronized with α-factor in G1 and further treated with 50 µg/ml phleomycin for 60 min, and then released from α-factor and phleomycin into YEPD as described in Materials and Methods. Aliquots of cells were collected at the indicated times after release from α-factor treatment and examined for DNA content by flow cytometry. (B) Rad53 phosphorylation after phleomycin treatment in G1 phase. Cells carrying YCp-RAD53-HA were arrested in G1 with α-factor and untreated or treated with 50 µg/ml phleomycin for 60 or 120 min maintaining the G1 arrest and then subjected to immunoblotting analysis. Strains used are wild-type (KSC1178), mec1Δ (KSC1186), tel1Δ (KSC1368), mec1-81 (KSC1400), mec1-81 tel1Δ (KSC1402).

Checkpoint responses after phleomycin treatment in S phase. (A) The S-phase checkpoint response to phleomycin. Cells were synchronized with α-factor in G1 and released in either the presence or the absence of 25 µg/ml phleomycin (PM). Aliquots of cells were collected at the indicated times after release from α-factor treatment and subjected to flow cytometric analyses as described in Materials and Methods. Dotted lines indicate the DNA content of 1C and 2C cells. The top panels represent asynchronous (As) cells not treated with phleomycin and are included as a reference. (B) Kinetics of Rad53 phosphorylation after release from G1 arrest into phleomycin. Cells carrying YCp-RAD53-HA were arrested in G1 with α-factor and released in medium containing 25 µg/ml phleomycin as in (A). Cells were harvested at time indicated and subjected to immunoblotting analysis. Strains used are wild-type (KSC1178), mec1Δ (KSC1186), tel1Δ (KSC1368), mec1-81 (KSC1400) and mec1-81 tel1Δ (KSC1402).

Protein kinase activity associated with Mec1 and Tel1. (A) In vitro phosphorylation of Rad53 and PHAS-1 by Mec1 and Tel1. Extracts were prepared from mec1Δ cells (KSC1186) carrying YEp-MEC1-HA, YEp-MEC1-KN-HA, or the control vector (left columns), or from tel1Δ (KSC1368) cells carrying YEp-TEL1-HA, YEp-TEL1-KN-HA or the control vector (right columns). Cells were grown to the mid-log phase and harvested for preparation of crude extracts. Extracts were subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitated HA-tagged Mec1 and Tel1 proteins were assayed for kinase activity using GST-Rad53C or PHAS-1 as a substrate as described in Materials and Methods. In each of the top three panels, ³²P incorporation into GST-Rad53C or PHAS-1 was detected by autoradiography. In each of the bottom panels, the amount of the Mec1 or Tel1 protein used for the kinase assay was examined by immunoblotting with anti-HA antibodies. (B) Substrate preference of Mec1 and Tel1. Extracts were prepared from MEC1-HA (KSC1212), TEL1-HA (KSC1517) and untagged (KSC006) cells. The kinase assay was performed with GST-Rad53C (2 µg) and PHAS-1 (0.6 µg), and ³²P incorporation was detected by autoradiography. In the bottom panel, the amount of the Mec1 and Tel1 protein used for the kinase assay was examined by immunoblotting. The star indicates bands which were detectable without addition of substrate (data not shown).